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PurifyingChallenging ProteinsPrinciples and Methods
GE Healthcare
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Purifying
Challenging ProteinsPrinciples and Methods
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2 Handbook 28-9095-31 AA
Content
Introduction...............................................................................................................................................................5
Challenging proteins ........................................................................................................................................................5
Outline ....................................................................................................................................................................................5
Common acronyms and abbreviations ..................................................................................................................6
Symbols .................................................................................................................................................................................7
Chapter 1Membrane proteins ..........................................................................................................................................................9
Introduction..........................................................................................................................................................................9
Classification of membrane proteins ....................................................................................................................10
Purification of integral membrane proteins for structural and functional studies .........................11
Starting material .............................................................................................................................................................12
Small-scale expression screening of histidine-tagged membrane proteins
from E. coli lysates .........................................................................................................................................................15
Cell harvest ......................................................................................................................................................................17
Cell disruption and membrane preparation ......................................................................................................18
Solubilization ....................................................................................................................................................................20
Purification ........................................................................................................................................................................29
Purification of non-tagged membrane proteins .............................................................................................38
Purity and homogeneity check ...............................................................................................................................40
Conditioning ......................................................................................................................................................................43
Proteomic analysis of membrane proteins ........................................................................................................45
References .........................................................................................................................................................................49
Chapter 2Multiprotein complexes ...............................................................................................................................................51
Introduction.......................................................................................................................................................................51
Pull-down assays ...........................................................................................................................................................53
Isolation of native complexes ...................................................................................................................................61
Isolation of recombinant protein complexes ....................................................................................................63
References .........................................................................................................................................................................67
Chapter 3Inclusion bodies ..............................................................................................................................................................69
Optimizing for soluble expression ..........................................................................................................................69
Strategies for handling inclusion bodies .............................................................................................................70Isolation of inclusion bodies ......................................................................................................................................71
Solubilization ....................................................................................................................................................................72
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Handbook 28-9095-31 AA 3
Refolding ...........................................................................................................................................................................72
Refolding using gel filtration .....................................................................................................................................74
Analysis of refolding .....................................................................................................................................................82
References .........................................................................................................................................................................82
Appendix 1Principles and standard conditions for different purification techniques ...........................................83
Affinity chromatography (AC) ...................................................................................................................................83
Ion exchange chromatography (IEX) ....................................................................................................................84
Hydrophobic interaction chromatography (HIC) ............................................................................................86
Gel filtration (GF) chromatography ........................................................................................................................87
Reversed phase chromatography (RPC) .............................................................................................................88
Appendix 2Manual and automated purification .....................................................................................................................89
Tagged recombinant proteins for simple purification ..................................................................................89
Manual purification techniques ...............................................................................................................................89
Automated purification using KTAdesign chromatography systems ................................................90
Appendix 3Column packing and preparation .........................................................................................................................93
Column selection ............................................................................................................................................................95
Appendix 4Conversion data: proteins, column pressures ..........................................................................................................96
Column pressures ..........................................................................................................................................................96
Appendix 5...................................................................................................................................................................97Converting from linear flow (cm/h) to volumetric flow rates (ml/min) and vice versa .....................97
Appendix 6Amino acids table ...........................................................................................................................................................98
Related literature.............................................................................................................................................. 100Ordering information .................................................................................................................................... 101
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IntroductionThis handbook is intended for students and experienced researchers with an interest in the
isolation of integral membrane proteins, multiprotein complexes, or in refolding proteins from
inclusion bodies. The aim is to present tools, strategies, and solutions available to meet the
purification challenges associated with these three classes of proteins.
For a background on techniques for protein purification in general and handling recombinant
proteins, we recommend the Recombinant Protein Purification Handbook and other
handbooks in this series (see Related literature on page 100).
Challenging proteinsOur knowledge and understanding of the structural and functional biology of soluble proteins
has increased dramatically over the last decade. Much of the technology for the production,
purification, and analysis of soluble proteins is now at a stage where generic purification
protocols allow relatively high success rates.
The situation is different for the areas that this handbook covers; integral membrane proteins,multiprotein complexes, and inclusion bodies. The need to handle and study these more
difficult groups of proteins is clear, given that:
about 30% of a typical cells proteins are membrane proteins, and more than 50% of the
current drugs on the market exert their actions via membrane proteins
while carrying out their enzymatic, structural, transporting, or regulatory functions,
proteins most often interact with each other, forming multiprotein complexes
a large proportion of normally soluble proteins that are overexpressed in E. coli end up as
incorrectly folded and insoluble protein in inclusion bodies
OutlineAfter a general introduction to each area, high-level consensus workflows are presented to
summarize current best practices in each area. Rather than providing a number of detailed
protocols that have been optimized for individual proteins, this handbook provides general
advice or generic protocols in a step-by-step format. The generic protocols are intended
as starting points for development of separation protocols. Details will typically have to be
changed to tailor the protocols for individual proteins. Furthermore, the required variations
to the generic protocols cannot be predicted and unless appropriate changes are made, the
protocols will only work poorly, if at allthis is one of the major challenges for the researcher
involved with these groups of proteins. To address this issue, the generic protocols are
presented with critical parameters identified, together with ranges of values to test for the
parameters. The handbook also provides guidance, hints, and tips when using protocols other
than those described here.
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Common acronyms and abbreviations
LTAB lauryl trimethylammoniumbromide
MBP maltose binding protein
MPa megaPascalM
rrelative molecular weight
MS mass spectrometryN/m column efficiency expressed
as theoretical plates per
meterPBS phosphate buffered salinepI isoelectric point, the pH
at which a protein has zeronet surface charge
PMSF phenylmethylsulfonyl fluoridepsi pounds per square inchPVDF polyvinylidene fluoride
r recombinant, as in rGSTRNase ribonucleaseRPC reversed phase
chromatographySDS sodium dodecyl sulfateSDS-PAGE sodium dodecyl sulfate
polyacrylamide gelelectrophoresis
TAP Tandem affinity purification
TCEP Tris (2-carboxyethyl)phosphine hydrochloride
TEV Tobacco etch virusu units (e.g., of an enzyme)Y2H Yeast-two-hybrid
A280
absorbance at specifiedwavelength (in thisexample, 280 nanometers)
AC affinity chromatographyBCA bicinchoninic acid
CBP calmodulin binding peptideCDNB 1-chloro-2-4-dinitrobenzeneC. elegans Caenorhabditis elegans
CF chromatofocusingCHAPS 3-[(3-cholamidopropyl)
dimethylammonio]-
1-propanesulfonateCMC critical micellar concentration
CV column volumeDAB 3,3-diaminobenzidineDDM dodecyl maltoside
DNase deoxyribonucleaseDS desalting (sometimes referred
to as buffer exchange)
DTT dithiothreitolE. coli Escherichia coliELISA enzyme-linked
immunosorbent assayFF Fast Flow
FW formula weight
GF gel filtration (sometimesreferred to as SEC: size
exclusion chromatography)GFP green fluorescent proteinGPCR G-protein coupled receptor
GSH reduced glutathioneGSSG oxidized glutathione
GST glutathione-S-transferaseGua-HCl guanidine-HClHIC hydrophobic interaction
chromatographyHMW high molecular weightHP High Performance
HRP horseradish peroxidaseIEX ion exchange
chromatography (also seen asIEC in the literature)
IMAC immobilized metal ion affinity
chromatographyIPTG isopropyl -D-thiogalactoside
LDAO lauryldimethylamine oxideLMW low molecular weight
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Symbols
This symbol indicates general advice to improve procedures or recommend action
under specific situations.
This symbol denotes mandatory advice and gives a warning when special care should
be taken.
This symbol highlights troubleshooting advice to help analyze and resolve difficulties.
Yellow highlights indicate chemicals, buffers, and equipment
Blue highlights indicate an experimental protocol
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Chapter 1Membrane proteins
IntroductionMembrane proteins play key roles in fundamental biological processes, such as transport ofmolecules, signaling, energy utilization, and maintenance of cell and tissue structures. About
30% of the genes determined by genome sequencing encode membrane proteins, and these
proteins comprise more than 50% of the current drug targets. Despite their importance, our
knowledge of the structure and function of membrane proteins at the molecular level lags far
behind that for soluble proteins. For instance, at the time of the publication of this handbook,
membrane proteins only represent around 1% of the 3-D atomic resolution structures that
have been deposited in the Protein Data Bank (http://www.pdb.org/).
Integral membrane proteins exist in a lipid environment of biological membranes
(biomembrane), but the available techniques for purifying, handling, and analyzing them
were optimized for water-soluble proteins in an aqueous environment. To be able to handle
and study membrane proteins they must be dispersed in an aqueous solution. This is usually
accomplished by adding a detergent that solubilizes the biomembrane and forms a soluble
complex with the lipids and membrane proteins (Fig 1.1). Solubilization is a harsh treatment
that has to be carefully optimized to avoid protein loss and inactivation. Protein denaturation
and/or aggregation are frequently encountered. Solubilization is one of the most critical
aspects in handling membrane proteins.
Other difficulties contribute to our lack of detailed structural and functional understanding of
membrane proteins. These include:
Low abundance. The quantity of membrane proteins is often very low in their naturalsetting. This makes their natural source impractical as a starting material for their
preparation.
Difficult production. Heterologous overexpression often results in low expression levels
and inactive protein due to insufficient membrane insertion and folding or lack of post-
translational modifications. Over-expression of membrane proteins can be toxic to the cell.
Fig 1.1. Schematic drawing of detergent solubilization of membrane proteins. Membrane proteins are transferred fromthe natural lipid bilayer (blue and yellow) to complexes with detergent (green) and, in some cases, lipids. A lipid-detergentmicelle, a detergent micelle, and free detergent are also shown.
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Membrane protein expression, purification, and analysis present considerable challenges.
Nevertheless, a substantial number of membrane proteins, especially from bacterial origin,
have been over-produced, isolated, and characterized in molecular detail. Also, several
studies aiming at mapping the membrane proteome in different organisms have been
published. Due to great efforts in a number of membrane protein labs, generic protocols for
membrane protein work have begun to emerge. These protocols are extremely useful as a
starting point in the lab. In the main part of this chapter, such protocols are provided togetherwith optimization advice and references for further reading.
Classification of membrane proteinsMembrane proteins are classified as peripheral or integral. Peripheral membrane proteins are
loosely associated with the membrane and are usually water soluble after being released
from the membrane. Peripheral membrane proteins generally present limited methodological
challenges when compared with integral membrane proteins. Throughout this handbook, the
term membrane protein refers to integral membrane protein unless otherwise indicated.
Integral membrane proteins are insoluble in water. They have one or more transmembrane
segments comprising polypeptide stretches that span the membrane. The transmembranemoiety can be constituted of a single or a bundle of-helices or of-barrel-like structures
composed of multiple polypeptide stretches. These proteins are called -helical membrane
proteins (Fig 1.2, left) and -barrel membrane proteins (Fig 1.2, right), respectively. The
-barrel membrane proteins are predominant in the outer membrane of Gram-negative
bacteria and mitochondria. Some proteins display both structures.
Fig 1.2. Three dimensional structure representations of an -helical membrane protein (left;Anabaena sensoryrhodopsin; PDB ID: 1XIO; [1]) and a -barrel membrane protein (right; ferric hydroxamate uptake receptor (fhua) fromE. coli; PDB ID: 1FCP; [2]). The structures are oriented such that the externally exposed area of each protein is at thetop. The yellow lines show the approximate locations of the lipid bilayer membrane. The yellow, horizontal lines are for
illustration purposes only and are not based on crystallographic data. Structures from The Protein Data Bank(http://www.pdb.org/).
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Purification of integral membrane proteins for structural andfunctional studiesThe high level workflow for the production and purification of integral membrane proteins
for structural and functional studies is shown in Figure 1.3. Each of the different stages in the
workflow is described in detail below, with relevant protocols. The protocols are intended as
starting points in the lab. Hints, tips, useful variations, and troubleshooting advice are also
given. The focus is on protocols for bacterial membrane production and purification sincethis is most common. Protocols for eukaryotic membrane proteins are less well developed.
However, much of the general advice is also valid for work with eukaryotic membrane
proteins.
Fig 1.3. Workflow overview for membrane protein isolation and purification for structural and functional studies.
Natural source Cloning and expression
Cell harvest
Expression screening
Cell disruption andmembrane prep
Solubilization
Purification
Purity andhomogeneity check
Conditioning
Structural and/orfunctional studies
Detergent screening
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Starting materialMembrane proteins from natural sourcesThe natural source of a membrane protein can be considered as a starting material for
purification. The only three-dimensional structure in molecular detail that has been reported
to date for a eukaryotic G-protein coupled receptor (GPCR), bovine rhodopsin, was obtained
with protein purified from bovine retina, where the protein is highly abundant (8). In many
cases, however, low abundance of the target protein precludes the use of the natural source
as starting material.
Examples of purifications from natural sources are presented later in this chapter.
CloningVectors used for the expression of soluble proteins are also commonly used for the
production of membrane proteins. It is useful to design a number (10 to 50) of different
constructs, including different homologues, to increase the chance that a particular
membrane protein can be produced in an active form.
In addition to the general considerations for choosing a vector (see Recombinant ProteinPurification Handbook, see Related literature, page 100), a number of other aspects relate
more specifically to choosing a vector for expressing membrane proteins.
Affinity tagging greatly facilitates expression screening based on chromatographic
enrichment, as well as optimization and use of protocols for purification of membrane
proteins. Polyhistidine tags are commonly used for membrane proteins, but the GST-
tag and others have also been used successfully. The insertion of a protease cleavage
site between the affinity tag and the target protein enables removal of the tag before
further analyses.
While a hexahistidine tag (His6) is the standard option for water-soluble proteins,longer histidine tags (with 8 or 10 histidine residues) are often used for membrane
proteins to increase the binding strength and thus improve yields in IMAC purification.
Drawbacks with longer (> 6 histidine residues) histidine tags are that expression
levels have been reported to be decreased in some cases and that a higher imidazole
concentration is required for elution.
Tags should generally be placed on the C-terminal end of the protein to reduce risk of
affecting the membrane insertion process based on the N-terminal signal peptide.
Fusion of the target membrane protein to a fluorescent protein tag such as GFP
in combination with a histidine tag allows direct and convenient visualization ofthe target during expression, solubilization, and purification and can speed up the
optimization of these processes (6).
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Expression and screeningTo correctly decide which conditions and constructs will be best suited for producing the
protein for the intended study, an efficient screening protocol is essential. Because of the
relatively low concentrations of overexpressed membrane proteins, it is useful to apply
affinity tags combined with separation methods that allow enrichment of the target protein.
Overexpression is a major bottleneck in the overall workflow for membrane protein
production. Several host systems are available and the final choice will depend both on
protein-specific requirements (e.g., for post-translational modifications) and practical aspects
(e.g., available equipment in the lab and expertise). It is often useful to try different hosts or
host strains in parallel for a particular target protein to increase the likelihood of success. In
addition, homologous membrane proteins from several sources can be cloned in parallel to
be able to select those that express well.
E. coli strain BL21 (DE3) is the most commonly used host for overexpression of membrane
proteins, in combination with a pET vector. High expression levels for functional membrane
proteins are usually more than an order of magnitude lower than for overexpression of
water-soluble proteins in E. coli. One inherent issue is that membrane proteins need to beinserted into membranes, and the availability of membrane structures in most cells is limited.
The issue with limited membrane availability can be addressed by using a host with large
amounts of internal membranes (e.g., Rhodobacter spp.; (3)). Another way of avoiding the
limitations set by available membranes is to produce the membrane protein as inclusion
bodies. This is usually not desired but may allow preparation of active protein through
solubilization of the inclusion bodies using denaturants followed by refolding. Successful
refolding of-barrel membrane proteins from inclusion bodies has been achieved (4) but
refolding of-helical membrane proteins is an even greater challenge. For a separate
discussion on inclusion bodies, see chapter 3.
A modest growth and expression rate is beneficial to avoid the formation of inclusion
bodies when using E. coli as a host. This can be achieved by the use of a weak
promoter, a low concentration of inducer and/or lowering the growth temperature
after induction.
An overview of different expression systems for membrane proteins is provided in Table 1.1.
For a review on important considerations for membrane protein expression, see reference 7.
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Table 1.1. Overexpression systems used for prokaryotic and eukaryotic membrane protein production
Expression system Advantages Disadvantages
E. coli The most widely usedoverexpression system for(prokaryotic) membrane protein
production.
Often not suitable for overexpression ofeukaryotic membrane proteins
No glycosylation and limited post-translational modifications
Yeast Can perform some post-translational modifications
Several different yeast systemshave been used for membraneprotein production (5)
Does not produce high cell densities(S. cerevisiae)
Hyperglycosylation can occur(S. cerevisiae)
Different lipids (compared with mammaliancells)
Insect cells Less complex growth
conditions compared withmammalian cells
Relatively high expressionlevels
Glycosylation
More costly and complex than E. coli or
yeast; different lipids (compared withmammalian cells)
Mammalian cells CHO, BHK and other cell linesare often used for functionalstudies of receptors
Authentic (mammalian) proteinis produced
High cost and complex work
Rhodobacterspp. High expression levels throughcoordinated synthesis offoreign membrane proteinswith synthesis of new internalmembranes (3)
Different lipids (compared with mammaliancells)
Cell free Allows expression of toxicproteins and proteins that areeasily degraded in vivo
Allows incorporation of labeledand non-natural amino acids.
High cost
Membrane protein insertion in membraneor detergent micelle has not been fullydeveloped
Disposable 96-well filter plates, from GE Healthcare, prepacked with affinity purification
media for histidine- or GST-tagged proteins can be used for reproducible, high-throughput
screening of protein expression. Typical applications include expression screening of different
constructs, screening for suitable detergents and solubility of proteins, and optimization of
the conditions for small-scale parallel purification. Plates are available prepacked with Ni
SepharoseTM High Performance or Ni Sepharose 6 Fast Flow for working with histidine-tagged
proteins (His MultiTrapTM HP or His MultiTrap FF, respectively); and Glutathione Sepharose 4Fast Flow or Glutathione Sepharose 4B for working with GST-tagged proteins (GST MultiTrap
FF or GST MultiTrap 4B, respectively).
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Small-scale expression screening of histidine-tagged membraneproteins from E. coli lysates
Cell lysis and solubilization
Buffer preparation
Lysis buffer: 20 mM sodium phosphate, 100 mM NaCl, 2 mM MgCl2, 20 mM imidazole, 0.5 mM Tris(2-carboxyethyl) phosphine hydrochloride (TCEP), 5 U/ml benzonase,1 mg/ml lysozyme, EDTA-free protease inhibitor cocktail, (concentration accordingto manufacturers recommendation),1-2% of a selection of detergents, pH 7.4
Procedure
1. Harvest cells from the culture by centrifugation at 7000 to 8000 g for 10 min or at 1000 to1500 g for 30 min at 4C.
2. Discard the supernatant. Place the bacterial pellet on ice.
3. Suspend the bacterial pellet by adding 5 to 10 ml of lysis buffer for each gram of wet cells.To prevent the binding of host cell proteins with exposed histidines, it is essential to includeimidazole at a low concentration in the sample and binding buffer.
4. Leave for 2 h with mild agitation at room temperature or 4C, depending on the sensitivity ofthe target protein.
5. Measure and adjust pH if needed.
Expression screening procedure
Materials
His MultiTrap HP or His MultiTrap FF
Centrifuge that handles 96-well plates
Buffer preparation
Binding buffer: 20 mM sodium phosphate, 500 mM NaCl, 20 to 40 mM imidazole, 0.5 mM TCEP,1 to 2% detergent, pH 7.4. (The optimal imidazole concentration is protein
dependent; 20 to 40 mM is suitable for many proteins.)
Wash buffer: 20 mM sodium phosphate, 500 mM NaCl, 20 to 40 mM imidazole, 0.5 mM TCEP,0.03% dodecyl maltoside (DDM), 1 to 2% detergent, pH 7.4,
Elution buffer: 20 mM sodium phosphate, 500 mM NaCl, 500 mM imidazole, 0.5 mM TCEP, 0.03%DDM, 1 to 2% detergent, pH 7.4
To increase the purity, use as high a concentration of imidazole as possible in the
sample and binding buffers without losing binding capacity.
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Preparing the filter plate
1. Peel off the bottom seal from the 96-well filter plate. Be sure to hold the filter plate over a sinkto accommodate any leakage of storage solution when removing the bottom seal.
2. Hold the filter plate upside down and gently shake it to dislodge any medium adhering to thetop seal. Return the filter plate to an upright position.
3. Place the filter plate against the bench surface and peel off the top seal.
4. Position the filter plate on top of a collection plate.
Note: Remember to change or empty the collection plate as necessary during the followingsteps.
5. Centrifuge the filter plate for 2 min at 200 g to remove the ethanol storage solution from themedium.
6. Add 500 l of deionized water to each well. Centrifuge the plate for 2 min at 200 g.
7. Add 500 l of binding buffer to each well to equilibrate the medium. Centrifuge for 2 min at200 g. Repeat once. The filter plate is now ready for use.
Blank run: Reducing agents may be used in sample and buffers. If this is the case,replace step 7 with the following steps:
7. Add 500 l of elution buffer/well. No reducing agent should be used in the elutionbuffer during this blank run. Centrifuge the plate for 2 min at 200 g
8. Add 500 l of binding buffer including reducing agent to each well to equilibratethe medium. Centrifuge for 2 min at 200 g. Repeat once. The filter plate is now
ready for use with reducing agent. Do not store His MultiTrap plates with buffers
containing reducing agents.
Centrifugation procedureCheck that all wells are drained after centrifugation. If not, then increase the
centrifugation force slightly.
Do not apply a force of more than 700 g during centrifugation.
1. Apply 100 l of lysate to each well of the filter plate and incubate for 3 min.
Note: If the yield of protein is very low, increase the incubation time and/or gently agitate thefilter plate to mix. The lysate volume can also be increased, and several aliquots of lysate canbe added successively to each well.
2. Centrifuge the plate at 100 g for 4 min or until all the wells are empty. Discard theflowthrough.
3. Add 50 l of binding buffer per well. Centrifuge at 200 g for 2 min.
4. Add 200 l of wash buffer per well. Centrifuge at 200 g for 2 min. Repeat twice.
5. Add 50 l of elution buffer per well and mix for 1 min.
Note: The volume of elution buffer can be varied (50 to 600 l per well), depending on theconcentration of target protein required.
6. Change the collection plate and centrifuge at 200 g for 2 min to collect the eluted protein.Repeat twice or until all the target protein has been eluted (A
280should be < 0.1, indicating that
all protein has been eluted).
Note: If necessary, change the collection plate between each elution to prevent unnecessarydilution of the target protein.
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The following detergents have been used with this protocol: 1% FOS-Choline 12,
1% undecyl maltoside, 1% dodecyl maltoside, 1% Cymal-5, 1% Cymal-6, 2% octyl
glucoside, 1% Triton X-100, 1% lauryl dimethylamine oxide (LDAO).
To optimize the protocol, vary the concentration of imidazole in the sample and in the
binding and wash buffers. A common variation range with His MultiTrap plates is 20 to
40 mM imidazole. If binding of the target protein is too low with these concentrations,
try 5 to 20 mM imidazole. In general, too low of an imidazole concentration in the
binding and wash buffers can cause adsorption of unwanted host proteins (and hence
a lower purity). Too high of an imidazole concentration can lead to a reduced yield of
the target protein.
Analysis
Samples can by analyzed by SDS-PAGE with Coomassie Blue staining (see Purity and
homogeneity check on page 40), or by dot-blot analysis on nitrocellulose membrane.
Histidine-tagged proteins can be detected using Anti-His Antibody.
Cell harvestMethods for cell harvest are host dependent and the same protocols are used for the
recovery of membrane proteins as for intracellular water-soluble proteins. Cell harvest of
suspension cultures is done by low speed centrifugation.
Cell harvest ofE. coli cultures
Buffer preparation
PBS: 10 mM phosphate, 2.7 mM KCl, 137 mM NaCl, pH 7.4
Centrifugation procedure
1. Centrifuge at 6000 to 9000 g at 4C for 15 min to collect the cells. Discard the supernatant.Resuspend the cells in 500 ml ice-cold PBS.
2. Centrifuge at 6000 to 9000 g at 4C for 15 min. Discard the supernatant. Resuspend the cellpellet in 10 ml ice-cold PBS, or another volume as required.
3. The resuspended cell pellet can be stored at 80C.
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Cell disruption and membrane preparationMethods for cell disruption are host dependent and essentially the same protocols are used
for recovery of membrane proteins as for water-soluble proteins. Cell disruption yields a
suspension of membrane fragments/vesicles that contains the membrane proteins. The
suspension also contains soluble proteins, remaining intact cells, various cell debris, and
other material as contaminants. These contaminants may need to be removed, depending on
the purification procedure used. Differential centrifugation is the standard approach for theisolation of membrane fragments/vesicles after cell disruption.
The pellet from cell harvest is resuspended in a suitable buffer for cell disruption (e.g., PBS).
DNase is added to reduce viscosity. It is useful to add a protease inhibitor cocktail to reduce
possible protein degradation. A selection of commonly used techniques for cell disruption are
summarized in Table 1.2.
Table 1.2. Overview of techniques for cell disruption to yield a suspension of membrane vesicles
Technique Principle Advantages (+) / Disadvanatges (-)
Liquid shear pressure(e.g., French press)
Rapid pressure drop by transferringthe sample from a chamber at highpressure through an orifice into achamber at low pressure
+ Fast and efficient, also for largevolumes
- Causes heating of the sample (cooling isrequired)
Ultrasonication Cells disrupted by high frequencysound
+ Simple- Causes heating of the sample, which
can be difficult to control by cooling- Proteins may be destroyed by shearing- Noisy
- Not for large volumes
Glass bead milling Agitation of the cells with fine glassbeads
+ Useful for cells that are more difficult todisrupt (e.g., yeast)
- Somewhat slow and noisy
Osmotic shock Change from high to low osmoticmedium
+ Simple, inexpensive- Only useful for disruption of cells with
less robust walls (e.g., animal cells)
Repeated freezing andthawing
Cells disrupted by repeatedformation of ice crystals; usuallycombined with enzymatic lysis
+ Simple, inexpensive+ Yields large membrane fragments- Slow
- May damage sensitive proteins anddissociate membrane protein complexes
- Low yield
Enzymatic lysis Often used in combination withother techniques, e.g., freeze-thawing or osmotic shock;lysozyme is commonly used tobreak cell walls of bacteria
+ Gentle+ Yields large membrane fragments- Slow- Low yield
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Cell disruption from frozen E. coli cell paste with overexpressed membraneprotein
Solutions
PBS: 10 mM phosphate, 2.7 mM KCl, 137 mM NaCl, pH 7.4
MgCl2: 1 M
Pefabloc: 100 mM
DNase: 20 mg/ml
Lysozyme: 10 mg/ml
Cell disruption
1. For each gram of cell paste combine, from the above solutions, 5 ml PBS, 5 l MgCl2, 50 l
Pefabloc, 5 l DNase, and 80 l lysozyme. Mix until the suspension is homogenous.
2. Sonicate on ice. Use the manufacturers recommended settings for amplitude and time for the
probe being used (e.g. 5 min of accumulated time; 9 s on, 5 s off).
3. Continue immediately with the membrane preparation.
This procedure was modified from reference 6.
Membrane preparation should be performed immediately after cell disruption.
Membrane preparation from E. coli.
All steps are carried out at 4C or on ice.
1. Centrifuge at 24 000 g for 12 min. Collect the supernatant.
2. Centrifuge the supernatant at 150 000 g for 45 min. Remove the supernatant and resuspendthe pellet in 10 ml PBS. The pellet contains the membrane fraction.
3. Centrifuge the resuspended pellet at 150 000 g for 45 min. Remove the supernatant andresuspend the pellet in 5 ml PBS.
4. Determine protein concentration using standard methods for soluble proteins such as theBiuret method or the bicinchoninic acid (BCA) method.
5. For storage, rapidly freeze the membrane suspension dropwise using liquid nitrogen and storeat 80C.
This procedure was taken from reference 6.
Different cell disruption protocols may give rise to different size fragments; the
centrifugation speed needs to be optimized accordingly.
Water crystals formed upon slow freezing may harm membrane proteins. Fast
freezing by submersion of the membrane suspension in liquid nitrogen forms
amorphous ice structures thus reducing the negative effects of freezing (some
researchers avoid freezing completely and always perform membrane protein
preparation from cell to pure protein as fast as possible, without interruptions; see
next point).
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For unstable membrane proteins, it may be beneficial to proceed directly with
purification after the preparation of membranes, and thus avoid freezing and storing
the membranes.
For small scale (0.5 to 50 ml) membrane preparations from E. coli, a procedure with
lysozyme treatment followed by water lysis and centrifugation is often efficient .
To facilitate protein purification, it can be useful to separate the inner and outermembranes from E. coli membrane preparations. This can be particularly helpful for
large (1 to >10 l) preparations. The inner membrane can be selectively solubilized
with 2% N-lauroylsarcosine (9). The outer membranes can then be recovered in
the pellet after a 1 h centrifugation. An alternative is to separate the inner and
outer membranes by a long (~10 h) sucrose gradient centrifugation, following cell
disruption.
It is sometimes possible to omit the fairly lengthy and cumbersome membrane preparation
step. The alternative is to first disrupt the cells and then directly solubilize membrane proteins
by the addition of detergent to the cell lysate, with no prior isolation of membranes. The
resulting solubilisate can be used for chromatography directly. By using chromatographycolumns that accept direct loading of unclarified homogenized cell lysate and detergent-
treated unclarified lysate (e.g., HisTrapTM FF crude columns), histidine-tagged membrane
proteins can be purified directly from the cell lysate (see Purification of histidine-tagged
membrane protein directly from crude, solubilized E. coli lysate, page 33).
SolubilizationThis is one of the most critical stages during the preparation of membrane proteins. During
the solubilization stage, membrane proteins are extracted from their natural environment,
the lipid membrane, to an aqueous environment by the use of detergents. Detergents act by
disintegrating the lipid bilayer while incorporating lipids and proteins in detergent micelles.
The hydrophobic surface areas of the membrane proteins and the lipid tails are buried in
the hydrophobic interior of the detergent micellar structures, while hydrophilic parts of the
proteins are in contact with the aqueous environment (Fig 1.1). An efficient solubilization
dissociates most lipid-protein and protein-protein interactions, thereby allowing the
separation of proteins.
The target protein can be purified in the presence of detergent by applying essentially any
of the existing protein purification techniques available for soluble proteins. A successful
solubilization protocol extracts the membrane protein at a high yield and results in stable
protein-detergent complexes (or protein-lipid-detergent complexes) where the protein retainsits active conformation.
Some membrane proteins require the interaction with native lipids from the lipid bilayer or
added exogenous lipids to remain in their active conformation. In such cases, it is essential
that the solubilization protocol enables the formation of a stable protein-lipid-detergent
complex and that it does not remove the required native lipid(s) associated with the target
protein. Harsh solubilization and purification procedures may lead to the removal of such
essential lipids, and hence inactivation of the protein.
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Detergents and CMCDetergents are amphipathic substances with a polar (hydrophilic) head group and a nonpolar,
(hydrophobic) tail. The polar part can be nonionic, anionic, cationic, or zwitterionic and
detergents are often classified accordingly (i.e., a detergent with an anionic polar part is
referred to as an anionic detergent). Advantages and disadvantages of the different classes of
detergents are listed in Table 1.3.
Table 1.3. Advantages and disadvantages with the different classes of detergents
Detergent class Advantages Disadvantages
Nonionic(e.g., dodecyl maltoside)
Generally mild and non-denaturingWidely used
May give low solubilization yields
Ionic (anionic or cationic)(e.g., SDS, LTAB)
Can be extremely efficient insolubilization
Often denaturingInterfere with ion exchangeseparations
Zwitterionic(e.g., FOS-Choline 12)
Often used in membrane proteincrystallization.Combines the advantages of ionic andnon-ionic detergents
More denaturing than nonionicdetergents
Above a certain concentration in an aqueous environment, detergent molecules associate
to form multimolecular complexes, micelles, with hydrophobic interiors and hydrophilic
surfaces. This concentration is referred to as the critical micellar concentration (CMC). The
CMC is different for different detergents and it also varies with pH, temperature and ionic
strength.
In general, all buffers and solutions used for membrane protein preparations (for
solubilization, purification, storage, etc.) should have a detergent concentration above
the CMC.
Several detergents that are recommended for solubilization of membrane proteins are listed
in Table 1.4. Chemical structures of a few example detergents from the different classes are
shown in Figure 1.4.
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22 Handbook 28-9095-31 AA
Table 1.4. Some recommended detergents for solubilization of membrane proteins
Detergent Class1 FWCMC2(mM)
Brij 35 N 1200 0.07
C12E8 N 539 0.11
CHAPS (3[(3-Cholamidopropyl) dimethylammonio]propanesulfonic acid)
Z 615 8
Cymal 7 (Cyclohexyl-n-heptyl--D-maltoside)
N 522 0.19
Decyl maltoside N 483 1.8
Digitonin N 1229 < 0.5
DDM (dodecyl maltoside) N 511 0.17
FOS-Choline 12 Z 352 0.12Hecameg (6-O-(N-Heptylcarbamoyl)methyl--D-gluco pyranoside)
N 335 19.5
LDAO (lauryldimethylamine oxide) Z 229 1
Nonidet P40 N 615 0.25
Nonyl glucoside N 306 6.5
Octyl glucoside N 292 18
Tween 20 N 1228 0.06
Triton X-100 N 647 0.23
1 N = nonionic; Z = zwitterionic2 At 20C to 25C and ~50 mM Na+
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Handbook 28-9095-31 AA 23
Fig 1.4. Chemical structures of selected detergents from the different classes.
Detergent screeningSeveral different detergents and conditions should be screened to establish the best
conditions for each membrane protein. Solubilization yield is monitored by assaying for the
target membrane protein in the solubilized fraction. In the protocol below, non-solubilized
material is removed by ultracentrifugation.
It is also possible to apply non-clarified solubilisate directly to specially designed
chromatography columns or multiplates that can handle cellular debris (see Expression
screening of histidine-tagged membrane proteins from E. coli lysates). Filtration can also be
used for clarification.
Protein detection may be performed by Western blot after SDS-PAGE. For low-expressed
proteins, a chromatographic enrichment step may be required before protein detection is
possible (see Conditioning on page 43). For highly expressed proteins, solubilization yield can
be estimated by SDS-PAGE with Coomassie Blue staining.
O O-
Na+
O
O
S
HO
HO
HO HO
O
O
N+
O-
Ionic: Sodium dodecyl sulfate
Non-ionic: Octyl glucoside
Zwitterionic: LDAO (Lauryl dimethylamine oxide)
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24 Handbook 28-9095-31 AA
In addition to determination of yield and homogeneity, it is often necessary to monitor
protein activity, which is the best indication of intact protein structure (and function). For
some membrane proteins, functional assays can be applied to the detergent-solubilized
protein. In other cases, reinsertion of the membrane protein into an artificial lipid bilayer
(membrane protein reconstitution) is necessary to perform a functional assay.
General detergent screening procedureBuffer preparation
PBS: 10 mM phosphate, 2.7 mM KCl, 137 mM NaCl, pH 7.4
Tris buffer: 20 mM at neutral pH
Table 1.5. Additives to PBS or Tris buffer for detergent screening
Membrane proteinconcentration(mg/ml)
Detergentconcentration (%) NaCl (mM) Temp(C) Time
Start condition 5 1(always above CMC)
100 4 2 h
Variation range 1 to 10 0.4 to 2(always above CMC)
100 to 500 4 to 37 5 min 5 h
For a list of recommended detergents, see Table 1.4.
Solubilization
1. Starting with the membrane preparation of known protein concentration, combine materialsas shown in Table 1.5 to give a total volume of 1 ml. Incubate with gentle mixing at the desiredtemperature for the time indicated.
2. Centrifuge at 100 000 g at 4C for 45 min.
3. Assay the supernatant for target protein. Generic assays that can be used are SDS-PAGE (seePurity and homogeneity check on page 40), rapid affinity purification followed by gel filtration(see Purity and homogeneity check on page 41) and affinity tag specific assays (e.g., a dot blotusing tag-specific antibodies). Also, assays for membrane protein activity should be considered.
The expression screening protocol on page 15, Small-scale expression screening
of histidine-tagged membrane proteins from E. coli lysates can be adapted to a
combined solubilization screening and binding screening protocol.
The results from a preliminary solubilization screening of EM29, an Mr29 000 histidine-tagged
membrane protein from E. coli, is shown in Fig 1.5. A strong band at the expected position for
the target protein indicates high expression and/or efficient solubilization. FC12, Triton X-100
and LDAO gave weaker bands than the other tested detergents. It was thus concluded that
these detergents were less suitable for solubilization of this protein. It should be stated alsothat high or reasonable yields must be combined with preservation of activity and stability
in detergent solution after solubilizationwhich is the next screening analysis that should be
considered.
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Handbook 28-9095-31 AA 25
Fig 1.5. Analysis by SDS-PAGE and Coomassie Blue staining of a combined expression screening and preliminarysolubilization screening of EM29. Eight different detergents were tested and elution was done in two steps (left and
right half of the gel, respectively). FC12 = FOS-Choline 12; UDM = undecyl maltoside; DDM = dodceyl maltoside; OG =octyl glucoside; TX-100 = Triton X-100; LDAO = lauryl dimethylamine oxide. Data kindly provided by Dr. Said Eshaghi,Karolinska Insitute, Stockholm, Sweden.
Specific lipids may be associated with the protein in the native membrane, and their
presence can be essential for protein activity. Retaining activity requires that these
lipids are still present after solubilization and purification. Harsh solubilization and
purification conditions can lead to removal of such lipids, resulting in inactivation of
the membrane protein.
DDM is often a good detergent to try in initial solubilization tests.CHAPS and digitonin have been reported to work particularly well for solubilization of
membrane proteins from Pichia pastoris.
FC12 DDM Cymal6 TX-100 FC12 DDM Cymal6 TX-100
UDM Cymal5 OG LDAO UDM Cymal5 OG LDAO
Mr 103
97
66
45
30
20.1
14.4
EM29
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26 Handbook 28-9095-31 AA
Solubilization screening for GST-tagged membrane proteins produced in E. coliThe method described below (Fig 1.6) provides a simple and rapid method to select for
optimal solubilization conditions to obtain the highest yield of GST-tagged membrane protein.
This screening procedure is based on the affinity of GST for Glutathione Sepharose 4B media.
The detergents selected for the screening must not affect the GST-binding activity
Fig 1.6. Detergent screening assay for a GST-tagged membrane protein.
Materials
GST Detection Module
GST SpinTrapTM Purification Module
GST MultiTrap 4B
Binding buffer: 10 mM phosphate, 2.7 mM KCl, 137 mM NaCl, pH 7.4 (PBS)Elution buffer: Binding buffer supplemented with 0.2% (w/v) detergent and 10 mM reduced
glutathione
1
Determine the detergenteffect on the enzymaticactivity of purified GSTwith the CDNB assay
Solubilize membranes indifferent detergents andconcentrations that donot affect the activity ofthe GST tag
Purify fusion protein using GSTSpinTrap Purification Module
Analyze yield by SDS-PAGE andactivity of membrane protein
(if assay is available)
96-well microplate
2
3
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Handbook 28-9095-31 AA 27
Solubilization screening
If the enzymatic activity of GST (as an indicator of GST binding activity) in the
presence of the selected detergents is unknown, evaluate GST activity in the
detergents using the CDNB assay provided in the GST Detection Module. Compare
GST activity in the presence and absence of each detergent. GST is known to be fully
active in DDM, CHAPS, octyl glucoside, Tween 20, Triton X-100, Brij35 and NP 40 at
detergent concentrations between 0.3 to 10 times CMC for each detergent .
1. Disrupt cells by lysozyme treatment combined with freeze-thawing and isolate membranes bycentrifugation at ~50,000 g for 30 min at 4C (see Membrane preparation from E. coli on page19 for additional information). Higher speed and longer centrifugation times (e.g., 100 000 gfor 60 min) may be required if more harsh cell disruption methods are used.
2. Solubilize the membrane pellet (1:10 w/v) in 5% (w/v) detergent solutions for 2 h on ice withmild agitation (see Detergent screening on page 23 for additional information).
3. Clarify by centrifugation at ~50 000 g for 30 min at 4C (or at 18 000g for 60 min at 4C).
4. Decant 500 l of the supernatant and purify with GST SpinTrap Purification Module or GSTMultiTrap 4Baccording to the supplied instructions. Elute with 500 l elution buffer. Assay forGST activity with GST Detection Module.
5. Analyze by SDS-PAGE (see Purity and homogeneity check on page 40).
This procedure was taken from reference 10.
Optimization of solubilization conditionsAfter establishing the initial conditions for solubilization, the conditions can be optimized by
further screening with one or a limited number of detergents. Useful screening parameters to
investigate are: ratio of protein concentration to detergent concentration. Protein concentrations in the
range of 1 to 10 mg/ml are typical
solubilization time and mixing conditions (e.g., mild agitation, end-over-end rotation, or
vigorous stirring)
pH
ionic strength
Size homogeneity can be used as an indicator of stability (and therefore optimum
solubilization conditions) because membrane proteins often oligomerize or aggregaterapidly when destabilized. Size homogeneity can be rapidly evaluated using SuperdexTM
200 5/150 GL (see Size homogeneity characterization on page 40). Figure 1.7 demonstrates
how Superdex 200 5/150 GL (column volume 3 ml) can be used to screen for homogeneity
under various pH and salt conditions.
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28 Handbook 28-9095-31 AA
Fig 1.7. Screening of pH and ion strength conditions for optimal homogeneity and stability of a detergent-proteincomplex. Rapid gel filtration with Superdex 200 5/150 GL showed a symmetrical peak when the separation wasperformed at pH 5.2 in 0.1 M NaCl (A), indicating a homogenous protein under these conditions. At somewhat highersalt concentration (D) a small peak appeared close to the void volume, indicating that oligomerization or aggregationappeared to a limited extent . At both pH 7.5 and pH 9.5 significant peaks were obtained close to the void volume,indicating severe oligomerization or aggregation. The complete screening procedure was achieved in only a few hours,including the time for column equilibration. Sample consumption was 6 10 l for the complete screen. Data kindly
provided by Dr. Said Eshagi, Karolinska Institute, Stockholm, Sweden.
Mixtures of detergents could also be considered.
Additives such as lipids and small amphiphiles (e.g., 1,2,3-heptanetriol) or known
ligands for the target protein may be useful to test. Small amphiphiles may affect
crystallization behavior by changing the size of detergent micelles. Ligands are
believed to reduce the dynamics of the protein structure and thus stabilize the protein
in detergent solution.
Solubilization for purificationAfter optimization, the solubilization protocol is performed at a scale that is appropriate for
the amount of membrane protein that needs to be obtained.
It is recommended to proceed with purification immediately after solubilization, to
minimize the loss of membrane protein activity due to aggregation, loss of structure,
or proteolytic degradation.
A detergent that works well for the solubilization of a particular membrane protein
may be less suited for other operations with the same protein (e.g., crystallization).
Procedures for exchanging detergents are described in Conditioning (see page 43).
For reviews on the use of detergents in membrane protein purification, see references 11 and
12.
0.0
5.0
10.0
15.0
20.0
mAU
0.0 0.5 1.0 1.5 2.0 2.5 3.0 ml
: .
-2.0
0.0
2.0
4.0
6.0
8.0
10.0
12.0
mAU
0.0 0.5 1.0 1.5 2.0 2.5 3.0 ml
:
: .
0.0
5.0
10.0
15.0
mAU
0.0 0.5 1.0 1.5 2.0 2.5 3.0 ml
: .: .
1.29
1.76
0.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
40.0mAU
0.0 0.5 1.0 1.5 2.0 2.5 3.0 ml
:
: .
0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
16.0
mAU
0.0 0.5 1.0 1.5 2.0 2.5 3.0 ml
: .
: .
1.29
1.78
0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0mAU
0.0 0.5 1.0 1.5 2.0 2.5 3.0 ml
.
1.3
: .
: .
A B C
D E F
0.1 M
0.3 M
pH 5.2 pH 7.5 pH 9.5
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Handbook 28-9095-31 AA 29
Solubilization with organic solventsSmall and stable membrane proteins are sometimes possible to extract from the lipid
bilayer in their active form by the use of organic solvents. For instance, a 110 amino acid
membrane protein was extracted from E. coli with a chloroform:methanol mixture, and initial
purification of the extract was done in this environment using an organic solvent resistant
chromatography medium and column (Fig 1.8; 13). For this membrane protein, a 1:1 ratio of
chloroform:methanol gave the best separation.
Fig 1.8. Gel filtration separation of solvent-extracted membrane proteins. Solid line = organic extract ofE. colimembranes in which EmrE (a multidrug resistance transporter protein) was overexpressed; broken line = organicextract of blank E. coli membranes. Two ratios of chloroform (C) and methanol (M) were used. The asterisk marks EmrEcontaining peaks. From reference 13. Used with permission. Copyright 2002 Elsevier Science (USA) Publication.
PurificationMembrane proteins are usually purified as protein-lipid-detergent complexes. The solubility
of the complexes in an aqueous environment allow the application of essentially the same
separation techniques as used for water-soluble proteins. The main difference is that the
purification of membrane proteins is carried out with detergent present in all solutions. This
is necessary because protein-detergent complexes are dynamic and would immediatelylose detergent molecules in the absence of free detergent. Detergent concentrations
should be above the CMC but can be kept about 10 times lower than what was used during
solubilization (typically in the 0.1% range).
0
50
100
150
200
250
A280
(mAU)
5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 ml
3:1 :M
1:1 C:M
Column: Sephadex LH-20 (40 ml bed volume) in 10 mm diameter column.Sample: Organic extract ofE. coli membranes in which EmrE (a multidrug
resistance transporter protein) was overexpressedEluent: Chloroform (C):methanol (M) 3:1 (upper) and C:M 1:1 (lower).Flow rate: 2 ml/minSystem: KTApurifier
*
*
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30 Handbook 28-9095-31 AA
High detergent concentrations can reduce the stability of the protein. However,
detergent concentrations need to be high during solubilization if the concentration
of membrane components is high. Once solubilization is completed, detergent
concentrations can be reduced. Detergents are often expensive, and it is also useful to
limit consumption for cost reasons.
Over-purification can lead to the removal of essential lipids from the protein-lipid-
detergent complex with concomitant loss of protein activity.
Membrane protein stability can often be improved by having 5% glycerol in all buffers
throughout the purification.
Purification of histidine-tagged membrane proteinsHistidine-tagged proteins have affinity for Ni2+ and several other metal ions that can be
immobilized on chromatographic media using chelating ligands. Consequently, a protein
containing a histidine tag will be selectively bound to metal-ion-charged media such as Ni
Sepharose High Performance (HP) and Ni Sepharose 6 Fast Flow (FF) while most other cellular
proteins will not bind or bind weakly. Elution is achieved by increasing the concentration ofimidazole. This chromatographic technique is often termed immobilized metal ion affinity
chromatography (IMAC).
Water-soluble, (histidine)6-tagged proteins are usually straightforward to purify following
standard protocols. Histidine-tagged membrane proteins are sometimes more problematic,
and weak binding to IMAC media is often reported. It has been speculated that this is
due to restricted accessibility of the histidine-tag for the IMAC ligand due to the binding
of detergent to the protein. To address the issue, longer histidine tags are routinely used
for overexpression of membrane proteins. Also, the presence of a linker, such as green
fluorescent protein (GFP), between the histidine tag and the target membrane protein has
been suggested to improve binding to IMAC media.
Historically, batch-wise purification has often been employed for the purification of
histidine-tagged membrane proteins. Batch-wise purification involves mixing the sample
with the chromatography media in an open vessel for a designated time, often overnight.
The suspension is then packed into a column for washing and elution of the bound protein.
Batch-wise purification can sometimes improve yields since adsorption times are longer than
for column separations. On the other hand, since a batch-wise procedure is longer it also
leaves the protein more exposed to proteolytic degradation or inactivation, and may thus
compromise the quality of the purified protein.
Purification of histidine-tagged membrane proteins can also be performed using column-based methods (see protocols on following pages).
Depending on the level of purity required for the final application, additional purification
steps can be performed after IMAC. For this purpose, gel filtration is possibly the most
common, and has the advantage that optimization is usually not needed. Anion exchange
chromatography can also be used and is often included between IMAC and gel filtration steps
(see the section Additional purification steps later in this chapter).
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Purification of histidine-tagged membrane protein from the solubilized, isolatedmembrane fraction
Materials
Column: HisTrap HP, 1 ml
Binding buffer: PBS, 20 mM imidazole, 0.1 to 1 % detergent (e.g., DDM), pH 7.4
Elution buffer: PBS, 0.5 M imidazole, 0.1 to 1% detergent (e.g., DDM), pH 7.4
Sample preparation
To 5 ml of solubilized membrane protein, add 0.20 ml of elution buffer to give a final imidazoleconcentration of 20 mM.
Purification
This purification procedure should be performed at 4C.
1. Fill the syringe or pump tubing with distilled water. Remove the stopper and connect thecolumn to the syringe (use the connector supplied), laboratory pump, or chromatographysystem drop to drop to avoid introducing air into the system.
2. Remove the snap-off end at the column outlet.
3. Wash out the ethanol with 3 to 5 column volumes (CV) of distilled water.
4. Equilibrate the column with 10 CV of binding buffer at a flow rate of 1 ml/min*
5. Apply the sample (using a syringe fitted to the Luer connector or by pumping it onto the
column) Use a flow rate of 1 ml/min.6. Wash with 10 CV of binding buffer at a flow rate of 1 ml/min.
7. Elute with a gradient of 0% to 75% elution buffer in 20 CV at a flow rate of 1 ml/min. When asyringe is used, elute stepwise with successively higher concentrations of imidazole.
8. After elution, wash the column with 5 CV 100% elution buffer followed by 5 CV binding buffer.
*One ml/min corresponds to approximately 30 drops/min when using a syringe with a HiTrap 1-
ml column. When using a larger column, a higher flow rate can be used. See column instructions.
The procedure can be scaled up by connecting two or three columns in series or by
using HisTrap HP 5 ml columns.
A relatively low NaCl concentration (e.g., PBS is 150 mM NaCl) is recommended
because membrane proteins tend to be less soluble at higher ionic strengths.
Higher concentrations (e.g., 300 to 500 mM NaCl) are often recommended for
IMAC of water-soluble proteins to reduce ionic interactions of contaminants with
the chromatographic medium. For some cases, even lower NaCl concentrations
(e.g., < 150 mM) should be applied for a membrane protein. Alternatively, the NaCl
concentration can be reduced directly after the IMAC step by desalting the material
using a HiTrapTM Desalting column.
It has been reported that by using gradient elution (with increasing concentrationsof imidazole) from an IMAC column, as in the protocol above, protein-lipid-detergent
complexes that differ only in lipid content can be separated.
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32 Handbook 28-9095-31 AA
Re-application of the flowthrough material, after step 5 above, to allow the sample to
pass through the IMAC column several times can be useful to maximize yield (14).
The yield can also be increased by decreasing the flow rate during sample loading.
For further purification, ion exchange chromatography and/or gel filtration is often
suitable (see Additional purification steps on page 35).
Figure 1.9 shows the purification of YedZ-TEV-GFP-(His)8 using the protocol above. YedZ isa transporter membrane protein from E. coli that was overexpressed in E. coli as a fusion
protein with GFP, a C-terminal (histidine)8
tag and a tobacco etch virus (TEV) protease
cleavage sequence.
Fig 1.9. Two-step purification ofE. coli YedZ-TEV-GFP-(His)8
from solubilized membranes prepared from E. coli cell culture.Fractions from (A) IMAC were further purified by (B) gel filtration. (C) SDS-PAGE analysis of selected fractions shows thepurity of the target protein. Fraction D from the HisTrap HP column was essentially homogeneous. M = molecular weightmarker. Peak E most likely contained light-scattering detergent-lipid aggregates. Data kindly provided by Dr. David Drew,Center for Biomembrane Research, Stockholm University, Stockholm, Sweden.
50 100 150 200 250 300 350
500
67
0
125
192
250
0
1000
1500
2000
2500
ml
mAU(280nm)
ImidazolemM
200 40 60 80
500
0
1000
1500
2000
2500
ml
mAU(280nm)
A BA
B
C
D
E
F
IMACColumn: HisTrap HP, 5 mlSample: Solubilized membranes with
YedZ-TEV-GFP-(His)8 fusion proteinSample load: 2.5 mg fusion protein/ml medium (after solubilization
at 3 mg/ml)Binding buffer: PBS, 0.1% DDM, pH 7.4Elution buffer: Binding buffer with 500 mM imidazole, pH 7.4Wash: 4% B over 20 CV, 425% over 20 CVElution: 50% BFlow rate: Load 0.3-0.5 ml/min, elution 1 ml/minSystem: KTAprimeTM
Gel filtrationColumn: Superdex 200 10/300 GLSample: Eluted fractions from HisTrap HPSample load: 0.5 mlBuffer: PBS, 0.1% DDM, pH 7.4Flow rate: 0.4 ml/minSystem: KTAprime
Totalm
emb
ranes
A B C D E FMr ( 103)
25015010075
50
37
25
20
C
M
YedZ-TEV-GFP-(His)8
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Purification of histidine-tagged membrane protein directly from crude,solubilized E. coli lysateThis protocol circumvents the need for membrane preparation and centrifugations. It uses
a chromatography column that was designed for the application of unclarified crude cell
lysate.
Material
Column: HisTrap FF crude, 1 ml
Binding buffer: PBS, 40 mM imidazole, 0.1% detergent (e.g., DDM), pH 7.4
Elution buffer: PBS, 1 M imidazole, 0.1% detergent (e.g., DDM), pH 7.4
Sample preparation
Suspend E. coli cell paste by addition of 5 ml PBS with 40 mM imidazole for each gram of paste.Perform cell lysis by lysozyme treatment and sonication (see Table 1.2). Add detergent to theunclarified lysate from a concentrated stock (e.g., DDM to a final concentration of 0.8%). Stir on ice
for 1.5 hours.
Purification
1. Fill the pump tubing or syringe with distilled water. Remove the stopper and connect thecolumn to the chromatography system tubing, syringe (use the provided Luer connector), orlaboratory pump drop to drop to avoid introducing air into the system.
2. Remove the snap-off end at the column outlet.
3. Wash out the ethanol with 3 to 5 CV of distilled water.
4. Equilibrate the column with 10 CV of binding buffer at a flow rate of 1 ml/min*
5. Apply the detergent-treated, unclarified lysate with a pump (0.5 ml/min) or syringe. Loadingvolumes of unclarified lysate are highly dependent on each specific sample.
*One ml/min corresponds to approximately 30 drops/min when using a syringe with a HiTrap1-ml column. When using a larger column, a higher flow rate can be used. See column
instructions.
Continuous, gentle stirring of the sample during sample loading is recommended
to prevent sedimentation. Sample loading at 4C may increase the viscosity of the
sample. An adverse effect of increased sample viscosity is that maximum back
pressure for the column is reached at a lower sample volume loading on the column.
Large volumes may increase back pressure, making the use of a syringe more
difficult.
6. Depending on the sample volume (larger sample volumes require larger wash volumes), washwith 10 to 30 CV of binding buffer at a flow rate of 1 ml/min.
7. Elute with a gradient of 0% to 12% elution buffer in 10 CV followed by 12% to 100% elutionbuffer in 5 to10 CV, all at a flow rate of 1 ml/min. When a syringe is used, elute stepwise withsuccessively higher concentrations of imidazole.
8. After elution, wash the column with 5 CV elution buffer followed by 5 CV binding buffer.
Figure 1.10 shows the purification of YedZ-TEV-GFP-(His)8
using the protocol above.
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34 Handbook 28-9095-31 AA
For further purification, ion exchange chromatography and gel filtration is often
suitable (see Additional purification steps on page 35).
The procedure can be scaled up by connecting two or three columns in series or by
using HisTrap FF crude 5 ml columns.
Fig 1.10. Purification of a (histidine)8-tagged membrane protein, YedZ-TEV-GFP-(His)
8directly from crude, solubilized E.
coli lysate using HisTrap FF crude, 1 ml. Peak fractions 14 and 15 were analyzed by SDS-PAGE. The gel was scanned todetect the GFP portion of the fusion protein. An arrow indicates the band corresponding to YedZ-TEV-GFP-(His)
8. In the
chromatogram, blue =A280
; orange = A425
(to detect YedZ); green = A485
(to detect GFP); gray = conductivity; red = % elutionbuffer. The overexpression vector was kindly provided by Dr J.-W. deGier, Centre for Biomembrane Research, Stockholm,Sweden.
800
200
400
600
mAU
00
20
40
60
80
% Elution buffer
80.0 85.0 90.0 95.0
14 15
100.0 ml
YedZ-TEV-GFP-(His)8
1514
Column: HisTrap FF crude, 1 mlSample: E. coli cell lysate, with overexpressed YedZ-TEV-GFP-(His)8 solubilized
in 0.8% dodecyl maltoside for 100 min on ice.Sample load: 50 mlBinding buffer: PBS, 40 mM imidazole, 0.1% DDM, pH 7.4Elution buffer: PBS, 1 M imidazole, 0.1% DDM, pH 7.4Flow rate: 1 ml/minGradient: 4% to 12% elution buffer in 10 ml; 12% to 100% elution buffer in 5 mlSystem: KTAexplorer
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Additional purification stepsFurther purification of the IMAC-purified, histidine-tagged membrane protein is usually
necessary for applications that require highly pure homogeneous material (e.g., for structural
characterizations). One or two additional chromatographic steps are usually sufficient. For
maximum efficiency, the purification scheme should be designed so that different separation
principles are utilized in the different steps.
A highly efficient purification scheme may consist of IMAC (separation according to specific
affinity) followed by desalting and ion exchange (separation according to charge differences)
and finally by gel filtration (separation according to size differences). Gel filtration is most
often used as the last step for the final removal of aggregates and for transfer of the sample
to a buffer suitable for further functional and structural studies. The separations are carried
out in the presence of detergent above the CMC (typically about 0.1%). Other conditions
are the same as for water-soluble proteins. Some suitable columns are listed in Table 1.6.
KTAxpress chromatography system can be used to automate multistep purifications
to produce highly pure proteins with minimum hands-on time. KTAdesignTM systems are
presented in Appendix 2.
High-resolution anion exchange can be used for both purification and characterization of
the charge homogeneity of purified membrane proteins. In most cases the ionic strength of
samples from IMAC must be reduced before application to ion exchange chromatography. Fig
1.14 shows charge characterization of an IMAC-purified membrane protein using Mono Q.
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36 Handbook 28-9095-31 AA
Table 1.6. Prepacked ion exchange and gel filtration columns for the additional purification of membrane proteins afterinitial IMAC purification
Technique Column Columnvolume
(ml)
Comment
Anion exchange Mono Q 5/50 GL 1 For the highest resolution and purity in
anion exchange chromatography at mgscale
Mini Q 0.24 or 0.8 For the highest resolution and purity inanion exchange chromatography at gscale
RESOURCE Q 1 or 6 A strong anion exchange medium; for highthroughput and easy scale up
HiTrap Q HP 1 or 5 A strong anion exchange medium; for fastseparations with high resolution and sharppeaks
HiLoad 16/10 QSepharose HP 20 Same as previous, but for larger sampleamounts
HiLoad 26/10 QSepharose HP
53 Same as previous, but for larger sampleamounts
HiTrap Q FF 1 or 5 A strong anion exchange medium; for fastseparations with good resolution
HiPrep 16/10 Q FF 20 Same as the previous, but for largersample amounts
HiTrap DEAE FF 1 or 5 Weak anion exchange medium; analternative to the strong Q anion
exchangers
HiPrep 16/10 DEAE FF 20 Same as the previous, but for largersample amounts
Cation exchange Mono S 5/50 GL 1 For the highest resolution and purity incation exchange chromatography at mgscale
Mini S 0.24 or 0.8 For the highest resolution and purity incation exchange chromatography at gscale
RESOURCE S 1 or 6 A strong cation exchange medium; for
high throughput and easy scale up
HiTrap SP HP 1 or 5 A cation exchange medium; for fastseparations with high resolution and sharppeaks
HiLoad SPSepharose HP
20 Same as previous, but for larger sampleamounts
HiLoad SPSepharose HP
53 Same as previous, but for larger sampleamounts
HiTrap SP FF 1 or 5 For fast separations with good resolution
HiPrep 16/10 SP FF 20 Same as the previous, but for largersample amounts
Table continued next page.
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Technique Column Columnvolume
(ml)
Comment
Gel filtration Superdex 200 10/300
GL
24 For highest resolution size separations of
membrane protein-detergent complexesin the molecular weight range 10 000 to600 000* in sample volumes up to 250 l
HiLoad 16/60Superdex 200 pg
HiLoad 26/60 Superdex200 pg
120
320
For size separations of proteins in themolecular weight range10 000 to 600 000* in sample volumes upto 5 ml (120 ml column) or 12 ml (320 mlcolumn)
Superdex 75 10/300 GL 24 For highest resolution size separations of proteins in the molecular weight range3000 -70 000* in sample volumes up to
250 lHiLoad 16/60 Superdex
75 pg
HiLoad 26/60 Superdex75 pg
120
320
For size separations of membrane protein-detergent complexes in the molecularweight range 3000 to 70 000* in samplevolumes up to 5 ml (120 ml column) or 12ml (320 ml column)
* Determined for soluble, globular proteins.
The solubility of membrane proteins can be very sensitive to ionic strength. When this
is the case, fractions eluted in a salt gradient from an ion exchange chromatography
column should be immediately diluted or run on a PD-10 Desalting, HiTrap Desalting,or a HiPrep 26/10 Desalting column to reduce the salt concentration (e.g., to
< 50 mM).
Gel filtration is excellent for the final purification step as it both removes aggregates
and simultaneously achieves buffer exchange, if required.
Tag cleavageThe affinity tag can be removed after purification if a protease cleavage site has been
inserted between the tag and the target protein. If using an KTAxpress system, on-column
cleavage protocols can be performed automatically. Protease activity can be affected by
the presence of detergents. Comprehensive data on the activity of different proteases indetergents is largely lacking, but both PreScission Protease and thrombin have been used
successfully with detergents. It is recommended to check for the extent of cleavage under
different conditions by SDS-PAGE (see Purity and homogeneity check on page 40).
PreScission protease exhibits excellent cleavage properties at 4C and is a useful
alternative for cleavage of sensitive proteins (15).
TEV protease is fully active in 9 mM decyl maltoside, but is partly inactive in several
other detergents (16).
See ordering information on page 103 for proteases available from GE Healthcare.
Table 1.6. continued
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38 Handbook 28-9095-31 AA
Purification of non-tagged membrane proteinsFor abundant membrane proteins, the natural source, rather than a heterologous host, is
often the best starting material for the purification. Due to the lack of an affinity tag, and
hence a highly specific initial purification step, a combination of several classical protein
purification techniques usually has to be employed to obtain sufficient purity. The columns
listed in Table 1.6 are also commonly used in purification schemes for non-tagged membrane
proteins, in combination with columns for other chromatography techniques. For anoverview, see the Protein Purification Handbook in this series.
Ion exchange chromatography is often used as the main purification step for the purification
of non-tagged membrane proteins. Figure 1.11 shows the anion exchange purification step
for a decyl maltoside solubilized glucose transporter membrane protein. The purity after this
step was above 90%.
Fig 1.11. Purification of the glucose transporter from human erythrocyte membranes (Glut1) by anion exchangechromatography. Erythrocyte membranes were solubilized with decyl maltoside. Glut1 eluted as a sharp peak at100 mM NaCl with a purity greater than 90%. From reference 17. Used with permission. Copyright 2001 ElsevierPublication.
Avoid using anionic detergents with anion exchange columns, and cationic detergents
with cation exchange columns.
As indicated earlier, gel filtration is the ideal final purification step for membrane proteins. It
is excellent for polishing as it removes aggregates and other impurities that are of a differentsize than the target protein, while simultaneously performing buffer exchange.
0.0
1.0
0.0
1.0
0 5 10 15
Concentration
NaCl(M)
Glut1
Elution volume (ml)
A280 nm
Column: HiTrap Q HP, 1 mlSample: Solubilized human erythrocyte membranes
Start buffer: 10 mM BisTris, 0.5 mM EDTA, 0.2% DM, pH 6.0Gradient: 0 to 500 mM NaCl in 15 CVFlow rate: Load: 0.5 ml/min; elution: 0.25 ml/min
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Figure 1.12 shows the use of repeated gel filtration for final purification of the bovine creatine
transporter from HEK293 cells (18). Membranes were solubilized with decyl maltoside and
initial purification was performed on a lectin column (wheat germ agglutinin immobilized on
Sepharose). Initial gel filtration on a Superdex 200 column gave a broad, nonsymmetrical
peak (Fig 1.12, upper). Additional gel filtration of a narrow fraction on the same column gave a
symmetrical peak, indicating homogeneity. Purity was confirmed by SDS-PAGE.
Fig 1.12.Purification of the bovine creatine transporter from HEK293 cells. Membranes were solubilized with decylmaltoside and initial purification was done on immobilized wheat germ agglutinin (not shown). (A) Material eluted fromthe WGASepharose column was concentrated and run on a Superdex 200 HR 10/30 column, which yielded multiplepeaks. (B) Material in the peak fractions (shown by the shaded bar) was concentrated. Re-chromatography of pooledfractions from this central region gave a symmetrical peak, indicating homogeneity. From reference 18. Used withpermission. Copyright 2005 Elsevier Scientific Publication.
-0.02
0.00
0.02
0.04
0.06
0.10
0.08
0.12
0.14
5 10 15 20 25
0.000
0.001
0.002
0.004
0.003
0.005
0.006
5 10 15 20 25
Volume (ml)
Volume (ml)
A
B
A280 nm
A280 nm
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40 Handbook 28-9095-31 AA
Purity and homogeneity checkPurity checkAs with water-soluble proteins, SDS-PAGE is the most widespread method for assessing
the purity of membrane proteins. Coomassie Blue, silver staining, or Deep PurpleTM (for
fluorescence) can be used for detection. The Laemmli system (19) is commonly used. Some
modifications may be necessary for membrane proteins, as outlined below. Suitable products
are listed in the ordering information section.
Boiling of the sample with SDS can cause aggregation of membrane proteins. As an
alternative to boiling, incubation at 60C for 30 min or at 37C for 60 min are useful
starting points for preparing the sample for SDS-PAGE. On the other hand, some
membrane proteins are fully compatible with boiling and boiling may be required for
complete solubilization with SDS.
Membrane proteins frequently do not move according to the expected molecular
weight in SDS-PAGE. They often move faster (i.e., appear smaller) possibly due to
incomplete unfolding or due to binding more SDS per mass unit protein as compared
with a water-soluble protein.
SDS-PAGE Clean-Up kit has successfully been used to treat samples containing
interfering detergent or that are too dilute for SDS-PAGE. With this kit, proteins are
quantitatively precipitated while interfering substances remain in solution.
Size homogeneity characterizationProtein aggregation is a common issue with membrane proteins. Aggregation often appears
to be irreversible and it may occur slowly over time but also rapidly and unexpectedly
with modest changes in ionic strength, pH, protein:detergent ratio and other factors. For
membrane proteins, it is as important to keep track of aggregation as it is to monitor proteinactivity.
Aggregation may not always be detected by SDS-PAGE since SDS solubilizes most
aggregates. Gel filtration is the method of choice for rapid detection of aggregation and it can
be applied under a wide variety of conditions. It is widely used as an efficient assay to assess
the size homogeneity in purified membrane protein samples.
Gel filtration allows detection of relatively small changes in the size of detergent-protein
complexes when different detergents are compared. Gel filtration is often used to give an
indication of the suitability of different detergents for a particular protein. Separation with
gel filtration is thus an important tool for qualifying the membrane protein preparation forfurther analysis. As an example, gel filtration is very often used for assessing the suitability
of different detergents for membrane protein crystallization. In some cases, however,